Effects of propionate and butyrate on chromatin accessibility

These modifications increase chromatin accessibility and have contrasting effects on normal and colorectal cancer cells, affecting growth, differentiation, and ion transport.

study: The short-chain fatty acid metabolites propionate and butyrate are unique epigenetic regulatory elements associated with diet, metabolism, and gene expression.. Image credit: Love Employee/Shutterstock.com

In a recent study published in natural metabolismA group of researchers investigated the effects of short-chain fatty acid (SCFA)-derived histone marks (propionylation and butyrylation) on chromatin accessibility and gene regulation in normal and colorectal cancer (CRC) cells and mouse intestine. I researched.

background

Histone post-translational modifications (PTMs) mediate the interaction between metabolism and histones. epigeneticsaffecting health and disease. beyond acetylationAcylation of short-chain lysines, such as propionylation (Kpr) and butyrylation (Kbu), links metabolism to gene regulation through acyl-coenzyme A (acyl-CoA) availability.

These modifications correlate with metabolic status and influence chromatin accessibility and transcription. SCFAs such as propionate and butyrate derived from dietary fiber metabolism exhibit antiproliferative effects in cancer by acting as substrates for histone acylation and altering chromatin structure.

Despite these findings, the precise mechanisms by which SCFAs regulate chromatin and gene expression remain unclear, and further studies are required to uncover their therapeutic potential.

About research

SW480 (from a primary adenocarcinoma of the colon), CCD841 (a non-tumorigenic colon epithelial cell line), and CT26 (a mouse colorectal cancer cell line) were cultured in appropriate media with supplements such as fetal bovine serum (FBS). I did. Penicillin-streptomycin.

Cells were maintained at a specific temperature (33°C for CCD841 and 37°C for others) in a humidified atmosphere containing 5% carbon dioxide (CO2). Treatments with sodium propionate (NaPr) and sodium butyrate (NaBu) were applied at different concentrations for 12 h and the cellular responses were evaluated. All cell lines were authenticated using short tandem repeat (STR) profiling and tested for mycoplasma contamination.

Histone proteins were extracted using an acid-based method and quantified using a bicinchoninic acid (BCA) assay. Equal concentrations were separated by polyacrylamide gel electrophoresis on gradient gels, transferred to nitrocellulose membranes, and analyzed by immunoblotting with primary and secondary antibodies.

Chemiluminescent signals were developed to identify histone modifications. ribonucleic acid (RNA)-mediated interference experiments used short hairpin RNA (shRNA) and complementary RNA. deoxyribonucleic acid (cDNA) to target histone acetyltransferase 1 (HAT1) and study its role in acetylation.

Cell viability was assessed using a fluorescence-based assay, highlighting the differential effects of NaPr, NaBu, and trichostatin A (TSA) treatments.

Histone acid extraction and mass spectrometry revealed dose-dependent histone modification by 13C-labeled NaPr. Perform chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin sequence (ATAC-seq), and under-targeted cleavage and tagmentation (CUT&Tag) assays to assess histone modifications and chromatin accessibility. We mapped gender.

Research results

H3K18 and H4K12 were selected for this study due to their known association with poor outcomes in CRC. To investigate SCFA-derived modifications, CRC cells were treated with physiologically relevant concentrations (0-10 mM) of NaPr and NaBu.

Using modification-specific antibodies, the presence of lysines Kpr and Kbu was detected on histones H3K18 and H4K12, and immunoblotting confirmed the dose-dependent deposition of these marks.

Mass spectrometry using isotope-labeled NaPr further verified that SCFA was directly incorporated as propionyl-CoA and butyryl-CoA to promote histone acylation. Of note, histone acetyltransferase 1 (HAT1) was suggested to be involved in mediating Kpr incorporation into H4K12, as depletion of HAT1 reduced this modification.

SCFA supplementation also affected intracellular acyl-CoA levels. Propionyl-CoA and butyryl-CoA levels increased dose-dependently in correlation with more advanced histone modifications, whereas acetyl-CoA levels were unchanged with propionate but decreased with butyrate supplementation.

These findings suggest that SCFAs alter the cellular acyl-CoA landscape and contribute to different epigenetic outcomes. Functional analysis demonstrated that Kpr and Kbu improve chromatin accessibility compared to acetylation (Kac), highlighting their role in transcriptional regulation.

Genome-wide analysis implicates propionate-derived H3K18pr and H4K12pr in specific regulatory regions, particularly in growth, differentiation, and signaling pathways such as transforming growth factor beta (TGF-β) and wingless/integrated and beta-catenin. It turns out that it is related to genes. Signal transduction pathway (Wnt/β-catenin).

Different binding patterns highlighted different transcription factor motifs, including Mothers Against Decapentaplegic Homolog 2/3 (SMAD2/3) and Jun. proto-oncogene B (junbu).

On the other hand, butyrate supplementation enriches genes that control cell motility, adhesion, and actin organization, leading to oncogenic loci such as myelocytomatosis proto-oncogene (MYC) and Fos proto-oncogene (FOS). It was shown to bind significantly.

In normal cells, Kbu marks were associated with adherens junctions and ion transport. In contrast, in CRC cells, CRC cells are associated with mesenchymal proliferation and apoptotic signaling, reflecting the contrasting roles of SCFAs in normal and cancer tissues.

Integrating ChIP-seq, ATAC-seq, and RNA-seq data highlighted the transcriptional effects of SCFA, demonstrating upregulation of differentiation-related genes and suppression of cell cycle regulators.

Histone modifications induced by SCFAs correlate with increased chromatin accessibility and transcriptional activation of target genes, supporting their potential as modulators of CRC epigenetics. Studies in mice confirmed the in vivo relevance of these findings, as dietary fiber metabolism caused similar chromatin changes in intestinal tissue.

conclusion

In summary, SCFAs such as propionate and butyrate are microbiome-derived metabolites with important epigenetic regulatory roles. In this study, we used histone post-translational modification profiling, ChIP-seq, CUT&Tag, ATAC-seq, and RNA-seq to identify the genomes affected by lysines Kpr and Kbu on histones H3 and H4 in CRC cells and normal cells. Areas and routes were identified.

SCFAs have been shown to increase chromatin accessibility, regulate oncogenic pathways such as Wnt/β-catenin and TGF-β, and alter the expression of genes such as MYC and FOS.

These findings suggest that SCFAs directly regulate gene expression and epithelial homeostasis and highlight the therapeutic potential of SCFAs in the prevention and treatment of colorectal cancer through dietary and microbial interventions.